Fiber scaffolds for use in esophageal prostheses

ABSTRACT

The development and construction of implantable artificial organs, and a process for manufacturing three-dimensional polymer microscale and nanoscale structures for use as scaffolds in the growth of biological structures such as hollow organs, luminal structures, or other structures within the body are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/385,612, filed Feb. 9, 2012, entitled “Fiber Scaffolds for Use inEsophageal Prostheses,” which claims priority to and benefit of U.S.Provisional Patent Application No. 61/466,039, filed Mar. 22, 2011,entitled “Electrospinning for Highly Aligned Nanofibers,” U.S.Provisional Patent Application No. 61/562,090, filed Nov. 21, 2011,entitled “Nanofiber Scaffolds for Biological Structures,” and U.S.Provisional Patent Application No. 61/585,869, filed Jan. 12, 2012,entitled “Biocompatible Nanofiber Materials for Biological Structures,”the entire contents of each of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The esophagus is an organ within the neck that permits travel of foodand saliva from the mouth to the stomach through peristalsis. It has agenerally tubular shape consisting of multiple layers ranging from amucosa layer on the lumen consisting primarily of epithelial cells to amuscular adventitia consisting primarily of smooth muscle cells,striated muscle cells and fibroblasts. The inner layer of muscle isoriented in a circumferential direction while the outer layer of muscleis oriented in a longitudinal direction (see FIG. 1). When at rest, theesophagus is nearly collapsed, but can expand to roughly 3 cm indiameter upon swallowing.

Peristalsis involves involuntary movements of the longitudinal andcircular muscles, primarily in the digestive tract but occasionally inother hollow tubes of the body, that occur in progressive wavelikecontractions. Peristaltic waves occur in the esophagus, stomach, andintestines. The waves can be short, local reflexes or long, continuouscontractions that travel the whole length of the organ, depending upontheir location and what initiates their action. In the esophagus,peristaltic waves begin at the upper portion of the tube and travel thewhole length, pushing food ahead of the wave into the stomach. Particlesof food left behind in the esophagus initiate secondary peristalticwaves that remove leftover substances. One wave travels the full lengthof the tube in about nine seconds. Peristaltic waves start as weakcontractions at the beginning of the stomach and progressively becomestronger as they near the distal stomach regions. The waves help to mixthe stomach contents and propel food to the small intestine. Usually,two to three waves are present at one time in different regions of thestomach, and about three waves occur each minute.

In the large intestine (or colon), the peristaltic wave, or massmovement, is continuous and progressive; it advances steadily toward theanal end of the tract, pushing waste material in front of the wave. Whenthese movements are vigorous enough to pass fecal masses into therectum, they are followed by the desire to defecate. If feces are passedto the rectum and not evacuated from the body, they are returned to thelast segment of the colon for longer storage by reverse peristalticwaves. Peristaltic waves are particularly important in helping to removegas from the large intestine and in controlling bacterial growth bymechanically acting as a cleansing agent that dislodges and removespotential colonies of bacteria.

Partial loss or complete loss of peristalsis due to the loss of theesophagus, small intestine and/or large intestine due to cancer or otherdiseases can have a catastrophic, if not fatal, effect on an animal. Anumber of in vivo prostheses for luminal structures such as theesophagus are known. Typically these prostheses are formed by donorstructures from cadavers or are manmade structures. However, theseexisting structures are subject to failure due to anastomotic stenosis,luminal stenosis, infection, dislocation, and migration, among othercauses. Therefore, there is an ongoing need for artificial or prostheticversions of organs such as the esophagus and intestinal tract that willprovide the patient, human or otherwise, with a functioning replacementfor the lost organ.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one photograph or drawingexecuted in color. Copies of this patent with color drawing(s) orphotograph(s) will be provided to the Patent and Trademark Office uponrequest and payment of necessary fee.

The accompanying drawings, which are incorporated into and form a partof the specification, schematically illustrate one or more exemplaryembodiments of the invention and, together with the general descriptiongiven above and detailed description given below, serve to explain theprinciples of the invention, and wherein:

FIG. 1A is an illustration of the anatomy of the esophagus from Frank H.Netter and FIG. 1B is an illustration of a portion of the smallintestine;

FIG. 2 is a photograph of an exemplary fiber deposition system, inaccordance with the present invention;

FIGS. 3A, 3B, 3C and 3D provide several photographs of examples ofrelatively small and large diameter tubes and irregular shapes derivedfrom electrospun fiber made with the process of the present invention;

FIGS. 4A, 4B, 4C and 4D provide a number of photographs illustrating thecontrol of cell orientation and differentiation based on discrete fiberalignment;

FIGS. 5A and 5B provide photographs of a 200 nm diameter fiber (on theleft) with pore sizes of a few microns and a 20 um diameter fiber (onthe right) with pore sizes of around 50 um; and

FIG. 6 is an SEM image of composite fiber scaffold that includes bothoriented fibers and random fibers.

DESCRIPTION OF THE INVENTION

Exemplary embodiments of the present invention are now described withreference to the Figures. Although the following detailed descriptioncontains many specifics for purposes of illustration, a person ofordinary skill in the art will appreciate that many variations andalterations to the following details are within the scope of theinvention. Accordingly, the following embodiments of the invention areset forth without any loss of generality to, and without imposinglimitations upon, the claimed invention.

The present invention relates generally to the development andconstruction of implantable artificial organs, and more specifically toa process for manufacturing three-dimensional polymer microscale andnanoscale structures for use as scaffolds in the growth of biologicalstructures such as hollow organs, luminal structures, or otherstructures within the body, particularly the esophagus (see FIG. 1A)and/or the small intestine, large intestine, duodenum, and jejunum.Exemplary versions of the manufacturing process of this inventioninclude preparing a preform that is based on an actual organ; electrospinning one or more layers of nanoscale (less than 1000 nanometers) ormicroscale (less than 50 microns) polymer fibers on the preform to forma nanofiber based scaffold. The fibers are typically formed byelectrospinning by extruding a polymer solution from a fiberization tip;creating an electronic field proximate to the fiberization tip; andpositioning a ground or opposite polarity within the preform. Thepreform may be rotated to align the fibers on the preform or a secondground or polarity may be placed in the preform and rapidly switchingthe electric field to align the fibers. The microscale and nanoscalepolymer fibers may be randomly aligned or maybe substantially parallelor both (see FIG. 6). These nanofiber structures may be seeded with oneor more types of biological cells prior to implantation in the body toincrease the rate of tissue growth into the scaffold. The scaffold mayinclude autologous or allogenic cells such as cord blood cells,embryonic stem cells, induced pluripotent cells, mesenchymal cells,placental cells, bone marrow derived cells, hematopoietic cell,epithelial cells, endothelial cells, fibroblasts, chondrocytes orcombinations thereof.

Choosing a material that accurately mimics the mechanical properties ofthe native esophagus (or other organ) can promote proper stem celldifferentiation and facilitate normal esophageal function such asperistalsis. Materials may be non-resorbable for permanent implantationor may be designed to slowly degrade while the host body rebuilds thenative tissue until the implanted prosthesis is completely resorbed.Permanent polymers may include polyurethane, polycarbonate, polyesterterephthalate and degradable materials may include polycaprolactone,polylactic acid, polyglycolic acid, gelatin, collagen, or fibronectin.The fibers may be electrospun onto a preform with the desired prosthesisshape (see FIG. 2). FIG. 2 is a photograph of an exemplary setup of a 5mm diameter rod with electrospun fiber being deposited onto the surface.The exemplary mandrel is coated with Teflon to facilitate removal of thescaffold after deposition or a slight taper (≈1°) can be manufacturedinto the mandrel. Nearly any size or shape can be produced from theelectrospun fibers by using a pre-shaped form and deposition method asshown in FIGS. 3A-3D.

Closely mimicking the structural aspects of the native esophagus (orother organ) is important with regard to replicating the function of thenative esophagus. By controlling the orientation of the fibers andassembling a composite structure of different materials and/or differentfiber orientations it is possible to control and direct cell orientationand differentiation (see FIGS. 4A-4D). Fiber orientation can be alteredin each layer of a composite or sandwich scaffold in addition to thematerial and porosity to most closely mimic the native tissue. Aproperly constructed scaffold will permit substantially completecellular penetration and uniform seeding for proper function andprevention of necrotic areas developing. If the fiber packing is toodense, then cells may not be able to penetrate or migrate from theexposed surfaces into the inner portions of the scaffold. However, ifthe fiber packing is not close enough, then the attached cells may notbe able to properly fill the voids, communicate and signal each otherand a complete tissue or organ may not be developed. Controlling fiberdiameter can be used to change scaffold porosity as the porosity scaleswith fiber diameter (see FIGS. 5A-5B). Alternatively, blends ofdifferent polymers may be electrospun together and one polymerpreferentially dissolved to increase scaffold porosity. The propertiesof the fibers can be controlled to optimize the fiber diameter, thefiber spacing or porosity, the morphology of each fiber such as theporosity of the fibers or the aspect ratio, varying the shape from roundto ribbon-like. The precursor solution described below may be controlledto optimize the modulus or other mechanical properties of each fiber,the fiber composition, the degradation rate (from rapidly biosoluable tobiopersitent. The fibers may also be formed as drug eluting fibers,anti-bacterial fibers or the fibers may be conductive fibers, radioopaque fibers to aid in positioning or locating the fibers in an x-ray,CT or other scan.

The effects of mechanical strain on electrospun polymer scaffolds hasbeen described in the literature (see, Microstructure-PropertyRelationships in a Tissue Engineering Scaffold, Johnson et al., Journalof Applied Polymer Science, Vol. 104, 2919-2927 (2007) and QuantitativeAnalysis of Complex Glioma Cell Migration on ElectrospunPolycaprolcatone Using Time-Lapse Microscopy, Johnson et al., TissueEngineering; Part C, Volume 15, Number 4, 531-540 (2009), which areincorporated by reference herein, in their entirety, for all purposes).Strains as low as 10% appear to rearrange and align the fibers in thedirection of loading. This alignment increases with the applied strainuntil over 60% of the fibers are aligned within ±10% of the direction ofapplied stress. If cells are present during fiber rearrangement in vivoor in vitro, they could conceivably be affected by these changesdepending on the overall rate of strain. Fiber alignment is retainedfollowing a single cycle of extension and release. This has significantbiological implications for a broad array of future tissue-engineeringoperations. As cells move across such a substrate, biased motion islikely as locomotion is based on forming and then dissolving a series offocal adhesions. Formation of these adhesions along the fiber directionmay be easier than those perpendicular to that direction although thiswill be partially controlled by the spacing between the fibers. This haslonger-term consequences for the eventual control of the architecture oftissues that develop upon such substrates.

Cellular mobility parallel to the fiber direction means that one couldconceivably control and direct cell proliferation and migration byprestraining scaffolds to align the fibers in certain directions. Thiscould result in tailored structures with highly aligned fibers and, as aresult, highly aligned cells. Of additional importance is the fact thatmany envisioned applications of tissue-engineering scaffolds willinvolve the use of cyclic stresses designed to achieve specificarchitectures in the biological component of the developing tissue. Ifthe scaffold experiences continuing hysteresis in which orientationincreases versus the number of cycles the efficiency of the overallprocess will be greatly enhanced. For blood vessels, as an example, theapplication of cyclic pressures will produce preferential stresses thatcould cause significant alignment of the fibers in the circumferentialdirection. This could cause cellular alignment in the circumferentialdirection, potentially creating a more biomimetic arrangement.

Within the context of this invention, electrospinning is driven by theapplication of a high voltage, typically between 0 and 30 kV, to adroplet of a polymer solution or melt at a flow rate between 0 and 50ml/h to create a condition of charge separation between two electrodesand within the polymer solution to produce a polymer jet. A typicalpolymer solution would consist of a polymer such as polycaprolactone,polystyrene, or polyethersulfone and a solvent such as1,1,1,3,3,3-Hexafluoro-2-propanol, N,N-Dimethylformamide, Acetone, orTetrahydrofuran in a concentration range of 1-50 wt %. As the jet ofpolymer solution travels toward the electrode it is elongated into smalldiameter fibers typically in the range of 0.1-30 am.

In preparing an exemplary scaffold, a polymer nanofiber precursorsolution is prepared by dissolving 2-30 wt % polyethylene terephthalate(PET) (Indorama Ventures) in a mixture of1,1,1,3,3,3-hexafluoroisopropanol and trifluoroacetic acid and thesolution is heated to 60° C. followed by continuous stirring to dissolvethe PET. The solution may be cooled to room temperature and the solutionplaced in a syringe with a blunt tip needle. The nanofibers are formedby electrospinning using a high voltage DC power supply set to 1 kV-40kV positive or negative polarity, a 5-30 cm tip-to-substrate distance,and a 1 μl/hr to 100 mL/hr flow rate. It is possible to use a needlearray of up to 1,000's of needles to increase output. Approximately0.2-3 mm thickness of randomly oriented and/or highly-aligned fibers maybe deposited onto the form, and polymer rings added, followed by anadditional approximately 0.2-3.0 mm of fiber added while the form isrotated. The scaffold may be placed in a vacuum overnight to ensureremoval of residual solvent (typically less than 10 ppm) and treatedusing a radio frequency gas plasma for 1 minute to make the fibers morehydrophilic and promote cell attachment.

In accordance with this invention, an exemplary preparation ofelectrospinning solution typically includes of polyethyleneterephthalate (PET), polycaprolactone (PCL), polylactic acid (PLA),polyglycolic acid (PGA), polyetherketoneketone (PEKK), polyurethane(PU), polycarbonate (PC), polyamide (Nylon), natural polymers such asfibronectin, collagen, gelatin, hyaluronic acid or combinations thereofthat are mixed with a solvent and dissolved. A form is prepared for thedeposition of nanofibers. Optionally, simulated cartilage or othersupportive tissue may be applied to the form and the fibers are thensprayed onto or transferred onto a form to build up the scaffold. Thepresent invention may be useful for the preparation of a number ofbodily tissues, including hollow organs, three-dimensional structureswithin the body such as trachea, esophagus, intestine or luminalstructures, such as nerves (epineurium or perineurium), veins andarteries (aorta, tunica externa, external elastic lamina, tunica medica,internal elastic lamina, tunica inima). Other preforms for mammals suchas primates, cats, dogs, horses and cattle may be produced.

While the present invention has been illustrated by the description ofexemplary embodiments thereof, and while the embodiments have beendescribed in certain detail, it is not the intention of the Applicant torestrict or in any way limit the scope of the appended claims to suchdetail. Additional advantages and modifications will readily appear tothose skilled in the art. Therefore, the invention in its broaderaspects is not limited to any of the specific details, representativedevices and methods, and/or illustrative examples shown and described.Accordingly, departures may be made from such details without departingfrom the spirit or scope of the applicant's general inventive concept.

For example, the present invention encompasses the following exemplaryembodiments and variants thereof: (i) a composite scaffold seeded withstem cells and promoted to differentiate into stratified tissue; (ii)separate scaffold layers or sheets seeded independently to formdifferent types of tissue and then assembled together using sutures,adhesive or welding to form a tubular shape and the stratified tissue;(iii) a scaffold implanted without cells for immediate replacement ofdamaged tissue and allow for cellular migration in vivo; (iv) anelectrospun fiber scaffold made from non-resorbable materials such aspolyethylene terephthalate, polyurethane, polycarbonate, poly etherketone ketone; (v) an electrospun fiber scaffold made from resorbablematerials such as polycaprolactone, polylactic acid, polyglycolic acid;(vi) an electrospun fiber scaffold made from natural polymers such ascollagen, gelatin, fibronectin, hyaluronic acid or any combination ofmaterial types; (vii) an electrospun fiber scaffold made from a singlelayer of oriented fibers or a composite comprising layers of orientedfiber to correspond to the native structure and help orient anddifferentiate cells (fiber orientation can be from a rotating mandrel(circumferential fiber alignment), a translating mandrel (longitudinalfiber alignment), or split ground method of using electrostatics toalign the fiber); (viii) using a pre-shaped mandrel or form to depositfibers onto to achieve a near net shaped esophagus or intestine or otherorgans that support/perform peristalsis—the pre-shaped form can have aslight taper machined into the mandrel or coated with a non-sticksurface to allow easy removal of the scaffold; and (ix) using apre-shaped mandrel or form to deposit fibers onto to achieve a near netshaped esophagus segment/patch or intestine segment/patch or other organsegment/patch that supports/performs peristalsis. A pre-shaped form canhave a slight taper machined into the mandrel or coated with a non-sticksurface to allow easy removal of the scaffold.

1. A fiber comprising an electrospun polymer and a radio opaquecompound.
 2. The fiber of claim 1, wherein the electrospun polymer isselected from the group consisting of polyethylene terephthalate,polycaprolactone, polylactic acid, polyglycolic acid,polyetherketoneketone, polyurethane, polycarbonate, polyamide,polystyrene, polyethersulfone, fibronectin, collagen, gelatin,hyaluronic acid, and combinations thereof.
 3. The fiber of claim 1,having a diameter of about 1000 nm or less.
 4. The fiber of claim 1,having a diameter of about 50 μm or less. 5.-20. (canceled)
 21. Thefiber of claim 1, formed into a layer having a fiber orientation and afiber spacing.
 22. The fiber of claim 22, wherein the fiber orientationis selected from the group consisting of substantially parallel,randomly oriented, and a combination thereof.
 23. The fiber of claim 22,wherein the fiber spacing is from about 2 am to about 50 am.
 24. Thefiber of claim 1, further comprising a plurality of biological cellsselected from the group consisting of cord blood cells, embryonic stemcells, induced pluripotent cells, mesenchymal cells, placental cells,bone marrow derived cells, hematopoietic cells, epithelial cells,endothelial cells, fibroblast cells, chondrocyte cells, and combinationsthereof.
 25. A method of fabricating a radio opaque fiber, the methodcomprising: depositing, by electrospinning at least one radio opaquefiber onto a preform; and removing the at least one radio opaque fiberfrom the preform.
 26. The method of claim 1, wherein the fiber has adiameter of about 1000 nm or less.
 27. The method of claim 1, whereinthe fiber has a diameter of about 50 am or less.
 28. The method of claim25, wherein electrospinning comprises: extruding a polymer solution froma fiberization tip; creating an electronic field proximate to thefiberization tip; and providing a ground or opposite polarity to thepreform.
 29. The method of claim 28, wherein the polymer solutioncomprises a polymer and one or more radio opaque compounds.
 30. Themethod of claim 29, wherein the polymer is selected from the groupconsisting of polyethylene terephthalate, polycaprolactone, polylacticacid, polyglycolic acid, polyetherketoneketone, polyurethane,polycarbonate, polyamide, polystyrene, polyethersulfone, fibronectin,collagen, gelatin, hyaluronic acid, and combinations thereof.
 31. Themethod of claim 25, wherein depositing the at least one radio opaquefiber comprises depositing a layer of radio opaque fibers, and whereinthe layer has a fiber orientation and a fiber spacing.
 32. The method ofclaim 31, wherein the fiber orientation is selected from the groupconsisting of substantially parallel, randomly oriented, and acombination thereof.
 33. The method of claim 31, wherein the fiberspacing is from about 2 μm to about 50 μm.
 34. The method of claim 31,further comprising subjecting the layer to a mechanical stress.
 35. Themethod of claim 31, further comprising seeding a plurality of biologicalcells onto the layer of radio opaque fibers.
 36. The method of claim 35,wherein the biological cells are selected from the group consisting ofcord blood cells, embryonic stem cells, induced pluripotent cells,mesenchymal cells, placental cells, bone marrow derived cells,hematopoietic cells, epithelial cells, endothelial cells, fibroblastcells, chondrocyte cells, and combinations thereof.